1. Field of the Invention
This invention relates to methods for developing novel, highly chemoprotectant crucifer germplasm. In particular, this invention relates to methods for developing novel, highly chemoprotectant broccoli and cauliflower germplasm. The invention relates to the development of germplasm which contains significant quantities of chemoprotective compounds that modulate mammalian enzymes which are involved in metabolism of carcinogens. These compounds induce the activity of Phase 2 enzymes, without inducing biologically significant activities of those Phase 1 enzymes that activate carcinogens. More specifically, the novel germplasm contains an enhanced alkyl/indole glucosinolate ratio.
This invention further provides a method of germinating cruciferous seeds and sprouts under conditions which increase antibiotic activity against a range of human pathogens, and which increase the chemoprotectant activity of the sprouts.
2. Description of the Related Art
It is widely recognized that diet plays a large role in controlling the risk of developing cancers and that increased consumption of fruits and vegetables reduces cancer incidence in humans. It is now believed that a major mechanism of protection depends on the presence of chemical components in plants that, when delivered to mammalian cells, elevate levels of Phase 2 enzymes that detoxify carcinogens.
Phase 2 enzymes are effective by detoxifying electrophilic forms of carcinogens which would otherwise damage DNA. Compounds which elevate the level of Phase 2 enzymes are termed xe2x80x9cselective inducers.xe2x80x9d Selective inducers of Phase 2 enzymes are designated monofunctional inducers. This means that they induce only Phase 2 enzymes, without significantly inducing Phase 1 enzyme activities. In contrast, compounds which induce both Phase 2 and Phase 1 enzymes are designated bifunctional inducers. (see Prochaska and Talalay (1988) Cancer Res. 48: 4776-4782). The monofunctional inducers are nearly all electrophiles and belong to at least 9 distinct chemical classes. (see Prestera et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2963-2969; Khachick et al. (1999) in ANTIOXIDANT FOOD SUPPLEMENTS IN HUMAN HEALTH, Packer, L. et al. (eds) pp 203-229, Academic Press, San Diego). The only apparent common property, shared by almost all of these inducers is their ability to react with thiol groups.
Monofunctional inducers are thus chemoprotective agents which reduce the susceptibility of mammals to the toxic and neoplastic effects of carcinogens. Chemoprotectors can be of plant origin or synthetic compounds. Synthetic analogs of naturally occurring inducers have also been generated and shown to block chemical carcinogenesis in animals. (see Posner et al. (1994) J. Med. Chem. 37: 170-176; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3147-3150; Zhang et al. (1994) Cancer Res. (Suppl) 54: 1976s-1981s).
Highly efficient methods have been developed for measuring the potency of plant extracts to increase or induce the activities of Phase 2 enzymes (Prochaska and Santamaria (1988) Anal. Biochem. 169: 328-336 and Prochaska et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2394-2398). In addition, these methods have been employed for isolating the compounds responsible for the inducer activities in plants and for evaluating the anticarcinogenic activities of these compounds and their synthetic analogs (Zhang et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2399-2403 and Posner et al. (1994) J. Med. Chem. 17: 170-176).
These methods have identified crucifer plants as a possible source of inducer activity. However, the levels of inducer activity among individual plants are highly variable, depending on the variety and on the growth and harvesting conditions.
It is now known that most of the inducer activity of crucifer plants is due to the presence and amounts of isothiocyanates and their biogenic precursors, glucosinolates. Glucosinolates are converted to isothiocyanates by the enzyme myrosinase, which is a thioglucoside glucohydrolase. Normally, myrosinase and glucosinolates are separated in the cell. If the cell is damaged, resulting in disruption of cellular compartmentalization, myrosinase comes into contact with glucosinolates, and converts them to isothiocyanates.
Although glucosinolates are not themselves inducers of mammalian Phase 2 enzymes, their conversion products, by virtue of myrosinase activity, are. Thus, it is the isothiocyanate products which are potent monofunctional inducers of Phase 2 enzymes in the murine hepatoma cell bioassay of QR activity.
However, not all glucosinolates produce isothiocyanates which are inducers of Phase 2 enzymes. Certain glucosinolates (e.g. alkylthioalkyl glucosinolates) produce isothiocyanates that are potent chemoprotective agents. Other glucosinolates (e.g. indole glucosinolates), produce compounds such as indole-3-carbinol and indole-3-acetonitrile, that are problematic for several reasons. First, such compounds are bifunctional inducers; that is, they induce both Phase 2 and Phase 1 enzymes. Phase 1 enzymes can activate xenobiotics thereby creating carcinogens. (Prochaska and Talalay (1988) Cancer Res. 48: 4776-4782). Second, the indole glucosinolates are only weak inducers of Phase 2 enzymes (Fahey J W et al. (1998) Chapter 2 in FUNCTIONAL FOODS FOR DISEASE PREVENTION I. Shibamoto T. et al. (eds), pp 16-22, ACS Symposium Series 701, Am Chem Soc, Washington D.C.). Third, these compounds themselves function as tumor promoters (Kim D J et al. (1997) Carcinogenesis 18(2):377-381). Finally, these compounds can form condensation products under the acid conditions encountered in the stomach, which are potent carcinogens very similar to dioxin (TCDD) (Bjeldanes L F et al. (1991) Proc Nat Acad Sci USA 88:9543-9547).
Thus, the amounts of inducer activity depends upon both the quality and quantity of glucosinolates present in crucifer plants. Market stage broccoli and cauliflower, for example, contain among the highest levels of the alkylthioalkyl glucosinolates, 4-methylsulfinylbutyl and 3-methylsulfinylpropyl glucosinolate, thus far identified in vegetables. They also contain, however, similar levels of the indole glucosinolates, glucobrassicin (indolyl-3-methyl glucosinolate), neoglucobrassicin, and 4-hydroxyglucobrassicin. Broccoli and cauliflower germinated seeds, sprouts, and young plantlets, on the other hand, contain higher concentrations of glucosinolates than do the market stage vegetable. The amount of glucosinolates present in the sprouts may depend to some extent upon the leakage of glucosinolates from the seeds upon imbibition and germination.
The processes of seed imbibition and germination, as well as priming, osmoconditioning, matri-conditioning and the like, though primarily associated with a net influx of water to the seed and seedling, also typically involve the leaking or leaching of chemicals from the germinating seed. The amount of chemicals leaking from the seed can be regulated by the milieu in which the seed is placed, although some leakage is inevitable. Furthermore, the amount of leakage may also be related to the quality of the seed lot, and to the type of seed.
The leachates of cruciferous seeds can exhibit potent antibiotic activity. This activity is effective not only against a range of human pathogens, but also against other microbes which commonly thrive or co-exist in commercial green sprout (such as bean sprout or green leafy sprout) production systems, which thus effectively contaminate these systems. The antibiotic activity of germinating crucifer seeds, in both the seeds and seedlings and the leachate resulting therefrom, is related to the glucosinolate content of the seed. While the leachates of alfalfa seed, the primary raw material of the green sprouts industry, actually stimulate the growth of Escherichia coli, leachates of cruciferous seeds (e.g., broccoli, cauliflower, kale, daikon, cabbage, arugula) contain glucosinolates and their isothiocyanate congeners (e.g., glucoraphanin and sulforaphane), which inhibit the growth of E. coli. Thus, glucoraphanin and its isothiocyanate congener, sulforaphane, not only induce Phase 2 enzymes of xenobiotic detoxification in mammals, and are chemoprotective, but they are also antimicrobial.
However, leakage during imbibition reduces the glucosinolate content of seeds and sprouts, thereby reducing their antibiotic and anti-carcinogenic activity. Furthermore, current commercial green sprout production systems result in only very low antibiotic activity of the leachate as well.
Thus, there is a need in the art to identify particular crucifer plants that yield high levels of Phase 2 enzyme-inducer activity for chemoprotection. There is also a need to identify crucifer plants that produce a known spectrum of specific inducers of Phase 2 enzyme activity in order to increase the efficiency with which specific carcinogens, or classes of carcinogens, are targeted for inactivation. Furthermore, there is a need to develop germplasm which comprise increased levels of Phase 2 inducer activity and to develop methods which effectively and efficiently manipulate the spectrum of inducers produced in particular varieties and lines. Finally, there is a need to obtain seeds and sprouts which exhibit decreased leakage of glucosinolates upon germination and sprouting. There is also a need to develop methods for commercial green sprout production which result in increased antibiotic production in the leachate without significantly decreasing the chemoprotectant activity present in the market sprout tissue.
It is therefore desirable to obtain crucifer varieties which possess consistent qualities and quantities of chemoprotectant activity. It is also desirable to obtain crucifer varieties which produce high levels of antibiotic activity during the sprouting process thereby reducing the risk the sprouts will become contaminated with human pathogens. It is also desirable to obtain crucifer varieties which also possess high quality chemoprotectant activity. Such crucifer varieties include open-pollinated and inbred broccoli and cauliflower lines which contain high levels of alkylthioalkyl glucosinolates relative to the levels of indole glucosinolates.
It is further desirable to obtain crucifer seeds and sprouts which exhibit decreased leakage of glucosinolates upon germination and sprouting. It is also desirable to obtain commercial green sprout production methods which result in increased antibiotic production in the leachate without significantly decreasing the chemoprotectant activity present in the market sprout tissuse.
Enhancing the anticancer qualities of cruciferous vegetables, seeds, and sprouts can be accomplished by the development of novel germplasm, using techniques of the instant invention.
It is therefore an object of the present invention to provide methods for developing novel crucifer germplasm, particularly broccoli and cauliflower germplasm, that contains significant quantities of,cancer chemoprotective compounds.
Another object of the present invention is to provide methods for developing novel crucifer germplasm, particularly broccoli and cauliflower germplasm, which contain substantial quantities of Phase 2 enzyme-inducers and are essentially free of Phase 1 enzyme-inducers.
It is a further object of the present invention to provide methods for developing crucifer germplasm, particularly broccoli and cauliflower germplasm, which contain substantial quantities of Phase 2 enzyme-inducing potential and non-toxic levels of indole glucosinolates and their breakdown products. Such germplasm contains an enhanced ratio of indole/alkyl glucosinolates in the appropriate tissue which is ultimately destined for the market and subsequent use. This tissue includes seeds, sprouts, and market-stage heads.
It is also an object of the present invention to provide methods for the creation of novel inbred or doubled haploid crucifer germplasm, particularly broccoli and cauliflower germplasm, by selecting for enhanced alkylthioalkyl/indole glucosinolate ratios.
It is also an object of the present invention to provide novel inbred or doubled haploid crucifer germplasm, particularly broccoli and cauliflower germplasm, which comprise enhanced alkylthioalkyl/indole glucosinolate ratios.
It is yet a further object of the present invention to provide methods for developing novel crucifer germplasm, particularly broccoli and cauliflower germplasm, with seeds of reduced leaching capacity and higher glucosinolate content.
It is also an object of the present invention to provide a method of germinating cruciferous seeds and sprouts under conditions which increase antibiotic activity against a range of human pathogens, and which increase the chemoprotectant activity of the sprouts.
These objects, and others, are achieved by providing a method of developing novel crucifer germplasm, particularly broccoli and cauliflower germplasm, with enhanced chemoprotectant properties, comprising (1) providing a population of doubled haploid plants, and (2) selecting an individual plant with an enhanced alkyl/indole glucosinolate ratio. Alternatively or in addition, the individual plant also produces seeds with reduced leakage of seed material upon germination and sprouting, resulting in higher glucosinolate content. The population of doubled haploid plants may be obtained from a parent plant by anther culture techniques.
The selecting step may comprise screening each plant to determine its alkyl/indole glucosinolate ratio. Furthermore, the screening step may comprise the steps of preparing an extract from a plant, and measuring the amount of alkyl and indole glucosinolates in said extract. Finally, the screening step may comprise screening the seed produced by each plant to determine the amount of leakage of material from the seed, and the glucosinolate content present in both the seeds themselves and in such leakage.
Also provided is a method for developing a novel inbred crucifer germplasm, particularly broccoli and cauliflower germplasm, with enhanced chemoprotectant properties, comprising (1) providing a population of doubled haploid plants, (2) selecting an individual plant with an enhanced alkyl/indole glucosinolate ratio, and (3) selfing the selected plant, and selecting the progeny from the selfed plant with an enhanced alkyl/indole glucosinolate ratio. Alternatively, or in addition, the individual plant also produces seeds of reduced leaching capacity resulting in higher glucosinolate content. Progeny from the selfed plant also produce seeds of reduced leakage of seed material.
Also provided is a method for creating novel inbred crucifer germplasm with enhanced chemoprotectant properties, comprising the steps of (1) providing a population of mutagenized plants, and (2) selecting an individual plant with an enhanced alkyl/indole glucosinolate ratio. Alternatively, or in addition, the individual plant also produces seeds of reduced leaching capacity resulting in higher glucosinolate content.
Also provided is a crucifer variety which comprises an enhanced alkyl/indole glucosinolate ratio, wherein said enhanced ratio is greater than about 10%, more preferably 20%, most preferably 30% or greater, of the ratio observed in the parent plant from which the variety is developed. Alternatively, or in addition, the crucifer variety produces seeds with reduced leakage of seed material upon germination and sprouting.
Also provided is a method of germinating cruciferous seeds and sprouts under conditions which increases antibiotic activity in the sprouting environment, and which increases the chemoprotectant activity of the sprouts, by imbibing and germinating the seeds in a ratio of water to dry seed which is just sufficient to permit normal growth of the sprouts over the normal commercial sprouting period and which does not result in any appreciable run-off from the sprouts.
Thus, the present invention is directed to a method for selecting a plant comprising measuring the ratio of alkyl glucosinolate to indole glucosinolate content in a tissue of a plant, and selecting the plant if the ratio is a specific number. Preferably, the plant is a double haploid plant such as one obtained by an anther culture technique, or a mutagenized plant such as a plant mutagenized with a chemical mutagen (e.g., ethylmethane sulfonate), wherein the plant is mutagenized before the measuring step.
Advantageously, the plant is a crucifer such as broccoli, preferably belonging to a cultivar selected from the group consisting of Saga, DeCicco, Everest, Emerald City, Packman, Corvet, Dandy Early, Emperor, Mariner, Green Comet, Green Valiant, Arcadia, Calabrese Caravel, Chancellor, Citation, Cruiser, Early Purple Sprouting Red Arrow, Eureka, Excelsior, Galleon, Ginga, Goliath, Green Duke, Greenbelt, Italian Sprouting, HiSierra, Late Purple Sprouting, Late Winter Sprouting White Star, Legend, Leprechaun, Marathon, Mariner, Minaret (Romanesco), Paragon, Patriot, Premium Crop, Rapine (Spring Raab), Rosalind, Salade (Fall Raab), Samurai, Shogun, Sprinter, Sultan, Taiko, and Trixie, and Viking.
Alternatively, the plant is a crucifer such as cauliflower, preferably belonging to a cultivar selected from the group consisting of Alverda, Amazing, Andes, Burgundy Queen, Candid Charm, Cashmere, Christmas White, Dominant, Elby, Snowball and selections of Snowball, Fremont, Incline, Milkyway Minuteman, Rushmore, S-207, Serrano, Sierra Nevada, Siria, Snow Crown, Snow Flake, Snow Grace, Snowbred, Solide, Taipan, Violet Queen, White Baron, White Bishop, White Contessa, White Corona, White Dove, White Flash, White Fox, White Knight, White Light, White Queen, White Rock, White Sails, White Summer, White Top, and Yukon.
The present invention is also directed to a method for producing a progeny plant comprising measuring the ratio of alkyl glucosinolate to indole glucosinolate content in a tissue of a potential parent plant, selecting the potential parent plant if the ratio is a specific number, and producing a progeny plant from the selected plant. Advantageously, the ratio of alkyl glucosinolate to indole glucosinolate in a tissue of the progeny plant is higher than the ratio of alkyl glucosinolate to indole glucosinolate in a tissue of the selected plant, and the tissue of the progeny plant and the tissue of the selected plant is of the same type. The present invention is also directed to a progeny plant produce by this method.
In preferred embodiments, the ratio in the tissue of the progeny plant is at least 10%, 20%, 30%, 50%, or 100% greater than the ratio in the tissue of the selected plant. In another preferred embodiment, the ratio in the tissue of the progeny plant is at least 10 times or 100 times as great as the ratio in the tissue of the selected plant. In yet another preferred embodiment, the ratio in the tissue of the progeny plant is between 10% greater than and 100% greater than, 20% greater than and 80% greater than, or 30% greater than and 60% greater than, the ratio in the tissue of the selected plant. In yet another preferred embodiment, the ratio in the tissue of the progeny plant is between 10 times as great and 100 times as great, or between 25 times as great and 75 times as great, as the ratio in the tissue of the selected plant. Advantageously, the progeny plant is produced by selfing the selected plant, or the progeny plant is a hybrid plant.
The present invention is also directed to a cruciferous plant, such as a broccoli plant or a cauliflower plant, wherein the ratio of alkyl glucosinolate to indole glucosinolate in a tissue of the plant is greater than 6, 10, 25, 50, 100, or 1000. In a preferred embodiment, the ratio of alkyl glucosinolate to indole glucosinolate in a tissue of the plant is between 6 and 1000, 10 and 500, 25 and 250, or 50 and 100.
The present invention is also directed to a method for selecting a seed or sprout comprising (a) adding a quantity of water to a seed or sprout, (b) measuring the concentration of glucosinolates in the leachate of the seed or sprout, and (c) selecting the seed or sprout if the concentration is a specific value.
The present invention is also directed to a method for producing a progeny plant comprising (a) adding a quantity of water to a seed or sprout, (b) measuring the concentration of a glucosinolate in the leachate of the seed or sprout, (c) selecting the seed or sprout if the concentration is a specific value, and (d) producing a progeny plant from the selected seed or sprout.
The present invention is also directed to a method for inhibiting or preventing the growth of a microbe in an environment capable of sustaining the microbial growth comprising administering to the environment a microbial growth-inhibiting amount of a cruciferous seed leachate or a cruciferous sprout leachate. Advantageously, the microbe is a bacterium such as Escherichia (e.g., Escherichia coli) or Staphylococcus (e.g., Staphylococcus aureus). Preferably, the leachate is a broccoli leachate or a cauliflower leachate. Ideally, the leachate contains a glucosinolate or an isothiocyanate.
The present invention is also directed to a method for preparing sprouts comprising (a) providing a quantity of seeds, (b) adding to the seeds between 0.6 and 1.0 g of water per g of seeds, (c) allowing the watered seeds to germinate and form sprouts, and (d) adding to the sprouts between 5 and 9 mg of water per sprout per 24 h for 24 to 120 h, thereby preparing the sprouts. Preferably, step (b) comprises adding to the seeds between 0.7 and 0.9 g (ideally 0.8 g) of water per g of seeds. Preferably, step (d) comprises adding to the sprouts between 0.6 and 0.8 mg (ideally 0.7 mg) of water per sprout per 24 h for 24 to 120 h. Preferably, step (d) comprises adding to the sprouts between 0.5 and 0.9 mg of water per sprout per 24 h for 48 to 120 h (ideally 72 h). Advantageously, the sprouts are cruciferous sprouts such as broccoli sprouts or cauliflower sprouts.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.